Ecodry premix with random primers

High-quality total RNA was extracted from the right hemisphere of the brain using RNeasy Universal Midi Kit (Qiagen) according to the manufacturer’s instructions. For Experiment 1, RNA was extracted from vehicle-treated mice and 667-COUMATE treated mice (n = 12 per group). For Experiment 2, RNA was extracted from mice administered 667-COUMATE with ziprasidone (0 mg/kg)(n = 11), mice administered 667-COUMATE with ziprasidone (0.3 mg/kg)(n = 11) and mice administered 667-COUMATE with ziprasidone (1.0 mg/kg)(n = 6). 20 μl cDNA solution was synthesised from 4 to 5 μg RNA using RNA-to-cDNA EcoDry Premix with random primers (Clontech), and was diluted 50-fold with distilled water. Quantitative PCR analysis was performed using the ΔC_t method using Gapdh, Hprt and Rn18 s as housekeeping genes (Trent et al., 2014 (link)) (primer sequences available in Supplemental Table S1).

3hr after behavioural testing, subjects were culled by cervical dislocation; whole brains were immediately removed, bisected sagitally, and frozen on dry ice. The 3hr post-behaviour timepoint was chosen in order to allow acute physiological changes induced by moderately aversive behavioural experiences to return to baseline. High-quality total RNA was extracted from the right hemisphere of the brain using RNeasy Plus Universal Midi Kit (Qiagen) according to the manufacturer’s instructions. For microarray analysis, three biological replicates per group were generated, with each replicate containing equal amounts of RNA pooled from four hemibrains; 260/280 absorbance ratios of 2.05-2.08 and RIN numbers of 9.4-9.8 were recorded for the six replicates using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). 20μl cDNA solution per hemibrain was synthesised from 4-5 μg RNA using RNA-to-cDNA EcoDry Premix with random primers (Clontech), and was diluted 50-fold with distilled water.